Abstract:

The invention relates to a composition comprising Leishmania promastigotes
or Leishmania amastigotes fractions or sub-fractions called "Fucose
Mannose Ligand" (FML) and saponin. The invention also comprises the use
of the composition for preparation of a blocking vaccine impeding
Leishmaniasis transmission in humans or animals.

4. Use of the composition of claim 1 for preparation of a blocking vaccine
to impede Leishmaniasis transmission in humans or animals.

5. Use of the composition of claim 1 for preparation of a blocking vaccine
to impede Leishmaniasis transmission consisting in administration of
Leishmania promastigotes or Leishmania amastigotes fractions or
sub-fractions called "Fucose Mannose Ligand" (FML) and saponin.

6. Use of the composition of claim 1 for preparation of reagents
comprising Leishmania promastigotes or Leishmania amastigotes fractions
or sub-fractions called "Fucose Mannose Ligand" (FML) and saponin, alone
or added to suitable pharmacology vehicles, for blocking Leishmaniasis
transmission in humans or animals.

8. Use of the composition of claim 2 for preparation of a blocking vaccine
to impede Leishmaniasis transmission in humans or animals.

9. Use of the composition of claim 2 for preparation of a blocking vaccine
to impede Leishmaniasis transmission consisting in administration of
Leishmania promastigotes or Leishmania amastigotes fractions or
sub-fractions called "Fucose Mannose Ligand" (FML) and saponin.

10. Use of the composition of claim 2 for preparation of reagents
comprising Leishmania promastigotes or Leishmania amastigotes fractions
or sub-fractions called "Fucose Mannose Ligand" (FML) and saponin, alone
or added to suitable pharmacology vehicles, for blocking Leishmaniasis
transmission in humans or animals.

Description:

FIELD OF INVENTION

[0001]This invention refers to pharmaceutical compositions suitable for
preparation of leishmaniasis transmission blocking vaccines in humans and
animals from Leishmania promastigotes or Leishmania amastigotes fractions
(FML). Such composition, when administered in combination with a saponin
adjuvant, blocks the visceral Leishmaniasis transmission in humans and
animals.

BACKGROUND OF THE INVENTION

[0002]In America and Mediterranean, the human visceral Leishmaniasis, or
kala-azar, is a canine zoonose. Phlebotominae insects when feeding from
wild canideous acquire the ethiologic agent, transmitting it later to
dogs. The subsequent transmission to humans by Phlebotominae insects
causes visceral human Leishmaniasis, a serious disease that can be fatal
if not adequately treated.

[0003]Approximately 1,000,000 new human occurrences of kala-azar are
registered annually in the world. A protective vaccine for prophylaxis
against human disease is not yet available. The best performance was
achieved with a first generation experimental vaccine which induced 12%
of protection, just among people that converted to positive in
intradermal tests for Leishmania lysate after complete vaccination. Even
today, the chemotherapy against kala-azar is highly toxicant and not
always effective.

[0004]Those vectors are a canine zoonose transmitted by insects and they
present infection reduction through ingestion of anti-Leishmania
antibodies present in serum of dogs submitted to protective vaccination
having great impact for reduction of the disease incidence in humans.

[0005]The brazilian patent PI9302386 describes a product called FML
antigen, "Ligante Fucose-Manose" or "Fucose Mannose Ligand" for
Leishmania donovani comprising a glycoprotein complex whose carbohydrate
portion presents 10% of Fucose and 47% of Manose, justifying the utilized
name. This complex strongly inhibits mouse peritoneal macrofags infection
due L. donovani promastigotes and amastigotes, it being species-specific
in the Leishmania gender. FML is strongly immunogenic in rabbits,
hamsters and mice stimulating intense antibodies production. It is
antigenic in humans and is recognized by high levels of specific
antibodies.

[0006]The FML initial biochemical analysis has revealed that FML is a
glycoprotein compound containing 29% of neutral sugar, 44% of protein,
11% of phosphate and hexamine traces composed by protein bands with
molecular weights ranging from 9 to 95 kDa, some protein bands having
carbohydrates: 36 and 54 to 68 kDa. The FML glycidic portion stands out
the presence of 10% of fucose and 47% of manose. It was also possible to
observe the presence of 30% of glucose, 12% of galactose and xylose in
trace-quantity.

[0007]The FML N-linked oligosaccharides form part of a ramified chain
alternating units of Manp 4-O, 3-O, Glc Nac 4-O-linked;
N-acetil-glucosamine as a ramification point and fucopyranose,
galactopyranose and manopyranose as terminal residues, or linear
oligosaccharides composed by units of Manp 4-O, 3-O and 2-O -linked,
2-O-linked, fucopyranose and galactopyranose as terminal residues (Thesis
presented at UFRJ in Apr. 14, 1998 by Robson R. Bernardo).

[0009]The Master's Degree Dissertation "Estudos sobre a glicoproteina GP36
de e sobre o eu potencial na vacinacao contra o calazar experimental"
("GP36 glycoprotein studies and its potential in vaccination against
experimental kala-azar") presented at UFRJ by Edilma Paraguai de Souza
identified a glycoprotein having molecular weight of 36 kDa as a FML
predominant antigen, called GP36. Leishmania donovani studies by C. B.
Palatnik de Sousa, H. S. Dutra, and R. Borojevic, in Acta Tropica,
53:59-72 confirm that GP36 is recognized by the hiper-immune rabbit serum
produced against total FML. Monoclonal antibodies obtained against FML
recognize predominantly GP36. The GP36 antigenicity was susceptible to
sodium periodate treatment indicating the glycidic nature of its epitopes
involved in the reaction with monoclonal antibodies, (ACE10 antibody),
therefore confirming its' glycoprotein nature. Furthermore, the Master's
Degree Dissertation presented at UFRJ in Mar. 25, 1998 by Edilma P. de
Souza shows that this fraction contains, among the amino acids, from 10
to 20% of aspartic acid, glutamic acid, alanine and leucine, from 5 to
10% of glycine, threonine, valine and lisin, from 5 to 2% of serine,
histidine, arginine, proline, tyrosine, methionine, isoleucine and
phenylalanine and that its N-linked oligosaccharides revealed fucose
residues with two substitution types (2,3-Me2-Fucose,
2,4-Me2-Fucose) and a predominance of manopyranose residues of
2,3,6,-Me3-Manose type, besides tri-Me3-Galactose corresponding
to 4-O-substituted manopyranose linear chains alternating with
substituted 3-O and 4-O fucopyranose residues.

[0010]The above mentioned R. Acta Tropica also reveals that does not exist
crossed reactivity between FML and glycoprotein 63, GP63, previously
isolated from Leishmania. Specific monoclonal antibodies L-3.2 and L-3.9
obtained by David Russell against GP63 do not develop any reaction to
FML, nor in Western Blotting assay, nor in ELISA assay, the latter
considered the most sensitive. This result is coherent and previsible,
considering the different extraction methods. The aqueous extraction of
FML, including double heating to 100° C., privileges the obtaining
of glycoconjugates whose glycidic fraction is resistant to these
conditions.

[0011]The GP36 protein portion was recently cloned in the E. coli. system
mentioned in "Molecular and Biochemical Parasitology", 120:315-319. It is
a nucleosidide hydrolase of 34 kDa L. (L.) donovani having 75% of
identity with Inosine Uridine Hydrolase (IUNH) of Crithidia fasciculata
and 90% of homology with rNH (IUH) of L. (L. major). The nucleoside
hydrolase of L. (L.) donovani is a specific diagnosis marker for canine
kala-azar, as described in this publication. The sequence of bases and
aminoacidics of the nucleoside hydrolase of L. (L.) donovani, described
in the Thesis of Doctorate "Clonagem molecular do antigeno GP36 de L.
donovani" ("Molecular cloning of L. donovani antigen GP36") presented at
UFRJ in May 14, 2002, is represented below:

[0012]A second FML antigenic component is a 55 kDa glycoprotein recognized
by 7 IgM type monoclonal antibodies and only one of the IgG type, as
mentioned in R. Acta Tropica.

DESCRIPTION OF THE INVENTION

[0013]The present invention describes the development of a prophylaxis
vaccine against canine visceral leishmaniasis based on L. donovani
saponin and FML antigen (Fucose Mannose ligand). The FML vaccine
developed 92-95% of specific protection (76-80% of vaccine effectiveness)
in Phase III assays against brazilian visceral Leishmaniasis, according
to studies described in Vaccine 19:1082-1092, 2001, and Vaccine
20:3277-3284, 2002. These studies showed that vaccination has reduced the
mortality and incidence of the canine disease. The research published in
Vaccine 22 (17-18):2234-2243, 2004 mentioned the immunotherapeutic effect
of the vaccine.

[0014]The term Transmission Blocking Vaccine (TBV) is used for
anti-malaria vaccines that stimulate the antibodies production in humans
against the sexual forms (gametes) of parasites present in the medium
intestine of the Anopheles vector. Mosquitos acquire those antibodies
during blood feeding, blocking the fertilization process and subsequent
development of the parasites in the medium intestine of the vector,
impeding the disease transmission by the insect.

[0015]Thus, the TBV is developed to increase the antibodies against the
gamete-phase of malaria parasite present in the mosquito intestine and in
spite of not eliminating the disease in the infected human, it prevents
the malaria propagation among the community.

[0016]The mosquito acquires the antibodies during blood feeding and they
would block the parasite development turning the vector in a
no-infectious vector. In case of malaria, the antigens that are
candidates to TBV are cellular surface antigens present in the
gamete-phase of the microorganism. A functional test is applied to the
analysis of the Phase I of this vaccine type. Mosquitos experimentally
created in laboratory and previously infected are fed with hiper-immune
serum against the subject antigen. Later the intestine of the mosquitos
can be dissected to determine the number of infectious gametocytes.

[0017]In the epidemic cycle of visceral leishmaniasis agents, Leishmania
chagasi and Leishmania infantum, the Phlebotominae, during blood meal,
ingests macrofags and monocytes containing amastigotes of infected dogs.
Those amastigotes delivered in the medium intestine of the vector differ
in flagellated and procyclical promastigotes linked to the medium
intestine epithelium. The procyclical promastigotes become divided by
metacyclogenesis acquiring virulence and they become metacyclic
promastigotes non-divided that are separated from the medium intestine
epithelium migrating to the buccal cavity and infecting new vertebrates
during the next blood meal.

[0018]The FML antigen is a surface antigen of L. donovani promastigotes
and amastigotes, highly immunogenic and protective in the canine
vaccination, so it could also be recognized by specific receivers in the
medium intestine of vectors as Lutzomyia longipalpis, acting as a
parasite ligand for the medium intestine membrane of the same.

[0019]In the present invention, assays were made relative to the FML
presence for specific receivers and to the growing capacity of the
antibodies in dogs after vaccination with Leishmune® (FML vaccine
under industrialized license) for blocking the adhesion of Leishmania
donovani and Leishmania chagasi procyclical promastigotes to the membrane
of the medium intestine of the vector, the "sandfly" Lutzomyia
longipalpis, making possible to characterize the potential blocking
effect for the FML vaccine.

[0020]The illustrations A, B, C and D are part of the present application
presenting the binding inhibition percentage, in percentage versus FML
concentration in μg/ml or anti-FML Fab antibodies in μg/ml for L.
donovani and L. chagasi.

[0022]Advantageously, the invention comprises a pharmaceutical composition
for preparation of a leishmaniasis transmission blocking vaccine in
humans and animals having Leishmania promastigotes or amastigotes
fractions called FML.

[0023]More advantageously, the invention refers to a Pharmaceutical
Composition having Leishmaniasis amastigotes or promastigotes fractions
called Fucose Mannose Ligand (FML) comprising native compounds containing
from 10 to 20% of glutamic acid, alanine, aspartic acid and glycine, from
5 to 10% of serine, histidine, lisin, threonine, proline and valine and
from 2 to 5% of arginine, methionine, isoleucine, tyrosine and
phenylalanine.

[0024]The invention comprises preferentially the use of the Pharmaceutical
Composition in the preparation of a blocking vaccine to impede the
Leishmaniasis transmission in humans or animals.

[0025]More preferentially, the invention comprises the use of the
Pharmaceutical Composition in the preparation of a blocking vaccine to
impede the Leishmaniasis transmission consisting in administration of
Leishmania promastigotes or amastigotes fractions called saponin-(FML).

[0026]The invention will be better understood from the following assay
having a merely exemplary and non-limitating scope.

[0027]Four healthy adult dogs without defined race were vaccinated with
Leishmune (licentiated industrialized vaccine). The animals received
three doses of the vaccine, with an interval of 20 days between each
dose, through subcutaneous way. The serums were collected 20 days after
the third application. The titles of the IgG type anti-FML antibodies
were determined using the FML-ELISA test. The IgG absorbence to 492 nm in
1/100 diluted serums were: 0.818; 0.761; 0.887 and 0.776. All serums
presented higher absorbence levels in the IgG2 than IgG1 subtype. To
purify the IgG samples, all samples were diluted, precipitated and
incubated by agitation. The precipitate was separated by centrifugation,
re-suspended and dialised. The isolated fraction was applied in a
Protein-to-Sepharose column. The non-linked fraction was eluted, while
the IgG purified fraction was recovered, neutralized and again dialised
in buffer.

[0028]The protein concentration was determined and IgG purified was
concentrated against polyethylene glycol. The Fab and Fc IgG fragments
were obtained using an Immuno Pure Fab kit and subsequent cromatography
in a Protein-to-Sepharose column. The Fab fragments were eluted in the
first 2 volumes of the column while the Fc fraction was removed using a
specific elution buffer.

[0029]Initially, the promastigotes binding to the medium intestine of the
vectors was inhibited in the presence of FML. For such case, intestinal
membranes of Lutzomyia longipalpis were dissected, longitudinally cut,
pre-incubated with FML (40, 100, 200 and 400 μg/ml) and later they
were incubated with 106 procyclical promastigotes.

[0030]The assays demonstrated the presence of FML receivers on the
membranes surface of the medium intestine of Phlebotominae. Such
receivers recognized the FML compound on the surface of L. donovani or L.
chagasi promastigotes phases acting as a parasite ligand for the medium
intestine membrane. The medium intestine membranes pre-incubation with
increased concentrations of FML then would inhibit the subsequent binding
of the promastigotes phases. The results are summarized in the
illustrations A and B. The FML concentration increase determines the
binding inhibition increase for L. donovani and L. chagasi.

[0032]Although the L. donovani isolated FML antigen has inhibited the
binding of both parasites, according to illustrations A and B, it was
observed that the inhibition was more pronounced against L. donovani
promastigotes than against L. chagasi indicating the species-specific
character of the homologs antigens recognition.

[0033]Binding inhibition assays were executed by pre-incubation of the
purified IgG Fab fraction from dogs vaccinated with Leishmune® (4, 40
and 400 μg/ml) with 106 procyclical promastigotes. Some minutes
after the interaction, the intestines were added and co-incubated. Later
the systems were individually washed and homogenized. The delivered
Leishmania promastigotes were counted in a Neubauer chamber. The results
represent the average and standard error of 2 experiments series with
7-10 membranes for each Fab fraction concentration.

[0034]The potential blocking of transmission by Leishmune vaccine in the
visceral canine leishmaniasis investigated in the above described assays
is summarized in the illustrations C and D. The analytic tests
demonstrated expressive differences among growing FML concentrations,
except between 4 and 40 μg/ml. Differences were not found between
binding inhibition for L. donovani or L. chagasi thus indicating that
antibodies induced by vaccinated dogs impede the binding of the
procyclical promastigotes phases of both parasites to the medium
intestine of the vectors, blocking and impeding the disease transmission.